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Biotinylation bovine gamma globulin-streptavidin ELISA (enzyme linked immunosorbent assay) plate and preparation method thereof

A gamma globulin and streptavidin technology, applied in the field of enzyme immunoassay, can solve the problems of unsatisfactory coating amount, long reaction time to reach equilibrium, low antibody utilization rate, etc.

Inactive Publication Date: 2012-12-05
天津中企华科生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The problem to be solved in the present invention is to provide a kind of pre-coated biotinylated bovine gamma globulin-streptavidin microplate and preparation method thereof, which can solve the above-mentioned defects brought by the traditional direct coating capture antibody ( The antibody utilization rate is low, the coating amount cannot meet the requirements, and the reaction reaches equilibrium time is long)

Method used

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  • Biotinylation bovine gamma globulin-streptavidin ELISA (enzyme linked immunosorbent assay) plate and preparation method thereof
  • Biotinylation bovine gamma globulin-streptavidin ELISA (enzyme linked immunosorbent assay) plate and preparation method thereof
  • Biotinylation bovine gamma globulin-streptavidin ELISA (enzyme linked immunosorbent assay) plate and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] One, a kind of ELISA plate of the present invention, the main raw material involved is as follows:

[0094] Ⅰ 96-well polystyrene microplate

[0095]96-well polystyrene microwell plate, transparent, domestic or imported, should have strong protein adsorption performance and good light transmission, preferably the product of Corning Company of the United States.

[0096] Ⅱ Preparation of biotin-labeled bovine gamma globulin

[0097] Bovine gamma globulin is a product of Sigma Corporation in the United States; activated biotin is a product of Sigma Corporation in the United States;

[0098] II streptavidin

[0099] Streptavidin was a product of Sigma, USA. When in use, prepare according to the volume of 15ml per 96 microwell plate (150 microliters / single microwell). Measure the volume of coating buffer-II = 15ml × pre-coating quantity (block), add streptavidin stock solution, adjust the protein concentration to 20 μg / ml to obtain streptavidin solution.

[0100] Ⅳ bov...

Embodiment 2

[0130] A diagnostic kit for erythropoietin ELISA, comprising the following components:

[0131] (1) ELISA plate prepared in Example 1: 96 wells (12 strips × 8 wells)

[0132] (2) Biotinylated EPO antibody: 6ml

[0133] (3) EPO enzyme-labeled antibody: 10ml

[0134] (4) Standard product: 6 bottles, 1.0ml in each bottle. The concentration is 0ng / ml, 0.5ng / ml, 2.5ng / ml, 5ng / ml, 10ng / ml, 30ng / ml

[0135] (5) Fixed value quality control serum: 2 bottles, 1.0ml per bottle

[0136] (6) Lotion: 20ml, 10 times concentrated, diluted with distilled water before use

[0137] (7) Chromogenic substrate A: 7.0ml.

[0138] (8) Chromogenic substrate B: 7.0ml.

[0139] (9) Stop solution: 7.0ml.

[0140] The preparation method of the kit is as follows:

[0141] (1) prepare the microtiter plate described in example 1

[0142] (2) Preparation of biotinylated EPO antibody

[0143] Raw material of antibody: EPO antibody - mouse anti-human erythropoietin monoclonal antibody, commercial reag...

Embodiment 3

[0172] A method for using the erythropoietin enzyme-linked immunoassay diagnostic kit prepared in Example 2, comprising the steps of:

[0173] (1) Take out all the components of the kit and prepare the serum samples to be tested, equilibrate to room temperature, and take out the microwell strips as required.

[0174] (2) Add 50 μl of standard, quality control serum, and specimen to the microwells, and then add 50 μl of biotinylated EPO antibody, shake for 10-20 seconds to mix, and bathe in 37°C water for 30 minutes;

[0175] (3) Remove the liquid in the micropores, wash 5 times with lotion, and pat dry on a clean towel.

[0176] (4) Add 100 μl EPO enzyme-labeled antibody to each well, shake for 10-20 seconds to mix well, and bathe in 37°C water bath for 30 minutes.

[0177] (5) Repeat step (3).

[0178] (6) Add 1 drop (50 μl) of chromogenic substrate A and chromogenic substrate B dropwise to each well, and place at room temperature (18-25° C.) for 15 minutes in the dark for ...

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Abstract

The invention provides a biotinylation bovine gamma globulin-streptavidin ELISA (enzyme linked immunosorbent assay) plate and a preparation method thereof. The biotinylation bovine gamma globulin-streptavidin ELISA plate comprises a polystyrene micropore plate, wherein the interior of each micropore of the polystyrene micropore plate is precoated with 1.5-3.0 micrograms of biotinylated bovine gamma globulin and 2.7-3.3 micrograms of streptavidin and is sealed by a sealing solution. By utilizing the ELISA plate provided by the invention, a capture antibody cannot be directly coated and is biotinylated, a biotin-avidin system can be used for combining the catch antibody with a solid-phase material (the micropore plate) in the to-be-detected antigen reaction process, thereby achieving the purpose of solid phase adsorption and separation and overcoming the defects that the antibody utilization ratio is low caused by a traditional method of directly coating the catch antibody, the coating amount cannot be met and the reaction equilibration time is retarded.

Description

technical field [0001] The invention belongs to enzyme immunoassay technology, in particular to a biotinylated bovine gamma globulin-streptavidin elisa plate and a preparation method thereof. Background technique [0002] Labeled immunoassay is a combination of immunoassay and tracer (labeling) technology. There is a complementary relationship in spatial structure between the antigen (Ag) and the corresponding antibody (antibody, Ab). When the two molecules meet, they will specifically combine to form an antigen-antibody complex (AgAb). Substances (radioisotopes, luminescent agents, etc.) are labeled on antigens or antibody molecules to form labeled antigens (Ag * ) or labeled antibody (Ab * ); In this way, the Ag can be detected by measuring the tracer substance (marker) * (Ab * ) or complex Ag * Ab(AgAb * ). Antigen-antibody reactions are highly specific, and tracer technology is highly sensitive. The perfect combination of the two endows labeled immunoassays with h...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N33/531G01N33/558G01N21/33
Inventor 王洪李会强张明华
Owner 天津中企华科生物科技发展有限公司
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